Soilborne plant diseases present an ongoing challenge to the plant pathologist. Plant pathogenic fungi cause severe economic losses throughout the world on many plant crops. For example, root rot of avocado (Persea americana Mill.) caused by Phytophthora cinnamomi Rands, is the major disease affecting avocado trees in California (Coffey, "Phytophthora root rot of avocado: an integrated approach to control in California" Plant Dis. 71:1046-1052 (1987)). Only two fungicides (metalaxyl and fosetyl-Al) are available that are only partially effective in suppressing the pathogen under conditions present in California (Cohen and Coffey, "Systemic fungicides and the control of oomycetes", Ann. Rev. Phytopathol. 24:311-338, (1986)). Total dependence on such site-specific fungicides is not recommended, because, aside from their high cost, fungicide-resistant strains of P. cinnamomi may occur through their excessive use (Cohen and Coffey, supra).
Another example is citrus postharvest disease caused by Penicillium digitatum. P. digitatum is a major source of decay of citrus fruit shipped from California and the Mediterranean region. This pathogen has developed fungicide resistance (Eckert and Brown, "Postharvest citrus diseases and their control" in "Fresh Citrus Fruits", Wardowski et al., Eds., pp. 315-361 (1986b)), rendering traditional methods of control ineffective.
An urgent need exists for an effective, broad-based, integrated approach to the management of such plant diseases. Traditional methods of control such as breeding for resistance, crop rotation and the use of chemical pesticides have been relatively unsuccessful and economically impractical.
"Biological control" involves the control of one organism by another antagonistic organism, defined as a biological agent with the potential to interfere in the life processes of plant pathogens. In the present invention "biological control" means the use of an organism to reduce the amount of inoculum or disease-producing ability of a plant pathogen. Mechanisms of biological control include 1) antibiosis, defined as the inhibition or destruction of the disease-causing organism by a metabolic product or products of the control organism, both specific and non-specific in nature, and including lytic agents, enzymes, volatile compounds or other toxic compounds, 2) competition, in which the introduced control organism successfully outcompetes the disease-causing organism for food and/or space, and 3) parasitism, in which the control organism parasitizes the disease-causing organism. Biological control may be accomplished by habitat management, for example to create an environment that favors organisms that are antagonistic to the pathogen; creation of host plant resistance to the pathogen; or by introduction of antagonists, or nonpathogenic strains. The term "biological control" as used herein may include antibiotic production and the effects of such production by the antagonistic organism in the suppression of the plant pathogen, however, the term as used herein includes the broader action of an antagonist in suppression beyond antibiotic production. Thus, demonstration of antibiotic production in vitro does not necessarily predict the ability of an organism to biologically control a plant pathogen in vivo, i.e. the ability to suppress infection by the pathogen of host plants present in soil. An antagonistic organism introduced into soil "in the field" must compete with other organisms and conditions present in the soil and on plant surfaces, including roots, stems, leaves and fruit to be effective in suppression of the pathogen. Antibiotic production alone may not be sufficient to cause suppression of the pathogen in the soil around the plant. Antibiosis often acts in concert with competition and/or parasitism. Therefore, the presence or absence of antibiotic production is not determinative of the ability of an organism to function as a biological control agent against a plant pathogen in soil (Fravel, "Role of Antibiosis in the Biocontrol of Plant Diseases", Ann. Rev. of Phytopathology 26:75-91 (1988)).
Attempts to use organisms such as bacteria and fungi to control plant diseases have been described. For example, Mishra et al. described the use of bacteria to control rust disease of wheat caused by Puccinia qraminis tritici (Mishra and Tewari, in "Microbiology of Aerial Plant Surfaces", Dickinson and Preece, eds., pp. 559-567, Academic Press, New York (1976).
Suppressive soils are defined as soils in which disease is minimal or diminishing even in the presence of susceptible host tissue, with or without reduction of the pathogen population. (Malajczuk, "Microbial antagonism to Phytophthora" in Phytophthora: Its Biology, Taxonomy, Ecology and Pathology, Erwin et al., Eds., Ch. 16 (1983)). Through introduction of single antagonistic organisms, repeated attempts have been made to enhance the suppressive effects of soils against specific plant pathogens (Ferguson, "Reducing plant disease with fungicidal soil treatment, pathogen free stock, and controlled microbial colonization", Ph.D. thesis, University of California, Berkeley (1958); Hadar et al., "Biological control of Rhizoctonia solani damping off with wheat bran culture of Trichoderma harzianum" Phytopathology 69:64-68 (1979); Papavizas and Lewis, "Physiological and biocontrol characteristics of stable mutants of Trichoderma viride resistant to MBC fungicides", Phytopathology 73:407-411 (1983); and Paulitz and Baker, "Biological control of Pythium damping-off of cucumbers with Pythium nunn: Influence of soil environment and organic amendments" Phytopathology 77:341-346 (1987)).
Species of Myrothecium, including M. roridum, are known to have strong cellulolytic activity which can be positively correlated with rhizosphere competence and strong competitive saprophytic ability (Ahmad and Baker, "Competitive saprophytic ability and cellulolytic activity of rhizosphere-competent mutants of Trichoderma harzianum" Phytopathology 77:358-362 (1987b); Domsch et al., "Myrothecium" in Compendium of Soil Fungi, Vol. 1, Domsch et al., eds., Academic Press, London, pp. 481-487 (1980)). Rhizosphere competence is defined as the ability to colonize the region of the root known as the rhizosphere, defined as the zone where maximum populations of soil organisms exist including the root surface, the adjacent soil and the external layer of the root itself (Russell, "Soil Conditions and Plant Growth", Chapter XII, in The Association between Plants and Microorganisms, Longmans, Pub., pp. 224-239 (1961)). M. roridum and other species of Myrothecium are strong antibiotic producers (Bamburg, "Biological and biochemical actions of trichothecene mycotoxins" Prog. Mol. Subcell. Biol. 8:41-110 (1983); Brian et al., "Production of antibiotics by species of Myrothecium" Mycologia 40:363-368 (1948); Jarvis et al., "Interaction between the antibiotic trichothecenes and the higher plant Baccharis megapotamica" Science 214:460-462 (1981); and Jarvis et al., "Macrocyclic trichothecene mycotoxins in Brazilian species of Baccharis" Phytopathology 77:980-984 (1987)). Myrothecium has been demonstrated to reduce root rot caused by Fusarium and Pythium in peas (Reyes and Dirks, Phytoprotection 66:23-29 (1985)).
A few attempts have been made to develop biological methods to control Phytophthora root rot of avocados in the field (Zentmyer, "Biological control of Phytophthora root rot of avocado with alfalfa meal", Phytopathology, 53:1383-1387 (1963)). Two avocado groves with soils suppressive to P. cinnamomi have been reported (Broadbent and Baker, "Behaviour of Phytophthora cinnamomi in soils suppressive and conducive to root rot" Aust. J. Agric. Res. 25:121-137 (1974a)). In this case, the continuous incorporation of large quantities of organic material and calcareous amendments partially restored some of the suppressive properties of the native rainforest soil, which naturally supports a microflora and microfauna that inhibits P. cinnamomi (Baker, "Biological control of Phytophthora cinnamomi", Proc. Int. Plant Propagators Soc. 28:72-79 (1978); Broadbent and Baker, supra (1974a); Cook and Baker, "The Nature and Practice of Biological Control of Plant Pathogens", American Phytopathological Society, St. Paul, Minn. 539 pp. (1983); and Rovira, "Organisms and mechanisms involved in some soils suppressive to soilborne plant diseases", in "Suppressive Soils and Plant Disease" Schneider, ed., American Phytopathological Society, St. Paul, Minn. 88 pp. (1982)).
Soils suppressive to Phytophthora have been described from several parts of the world, however, the nature of such suppression is still largely unresolved (Cook and Baker, supra; Scher et al., "Mechanism of biological control in a Fusarium-suppressive soil", Phytopathology, 70:412-417 (1980)). Generally, no single physical, chemical, or biological property of a soil has been identified as a primary determinant in soils suppressive to Phytophthora. No single organism has been isolated that could completely reproduce the suppression (Broadbent and Baker, "Association of bacteria with sporangium formation and breakdown of sporangia in Phytophthora", Aust. J. Agric. Res. 25:139-145 (1974)). Additionally, the suppressive phenomenon was not transferable to new soils (Cook and Baker, supra), as is the case with some soils suppressive to Fusarium (Scher and Baker, supra).
M. roridum together with another species (M. verrucaria) are well documented as soil microorganisms and have a cosmopolitan distribution (Domsch et al., "Myrothecium", pages 481-487 in: Compendium of Soil Fungi, Vol 1, Academic Press (1980); Tulloch, "The Genus Myrothecium Tode ex Fr.," Mycol. Pap. 130:1-42 (1972)). They are found in a wide range of soils, especially those high in organic matter, and on a number of other substrates (Boland et al., "Biological control of Sclerotinia sclerotiorum with fungi isolated from petals" (Abstr.) Phytopathology, 77:115 (1987); Domsch et al., supra; Thode, "Fusarium solani, Fusarium equiseti and Myrothecium verrucaria as parasites of Pythium ultimum", M. S. Thesis, University of California, Davis, (1981); Tulloch, supra).
Pathogens such as P. cinnamomi, which attack the roots of their hosts and grow internally until the plant is killed, are difficult to control once penetration of the host root has occurred. Consequently, promising biological control organisms may need to be rhizosphere-competent (Ahmad and Baker, "Rhizosphere competence of Trichoderma harzianum, Phytopathology 77:182-189 (1987a); Ahmad and Baker, (1987b)) to antagonize the pathogen before infection.
For biological control, an inoculum is used consisting of an aqueous suspension containing the antagonistic organism to be introduced into the soil, or onto a plant surface to fight the pathogen. For applications onto a plant surface, the aqueous suspension may be sprayed or, for applications to certain plant parts such as fruits and tubers, the parts may be dipped in the inoculum. Bran such as wheat bran is an effective and economic food base for introducing specific biological control agents into soils (Hadar et al., "Biological control of Rhizoctonia solani damping off with wheat bran culture of Trichoderma harzianum", Phytopathology 69:64-68 (1979); Lewis and Papavizas, "A new approach to stimulate population proliferation of Trichoderma species and other potential biocontrol fungi introduced into natural soils", Phytopathology 74:1240-1244 (1984); Lewis et al., "Effect of mycelial preparations of Trichoderma and Gliocladium on populations of Rhizoctonia solani and the incidence of damping-off", Phytopathology 75:812-817 (1985); Papavizas and Lewis, "Introduction and augmentation of microbial antagonists for the control of soilborne plant pathogens", pages 305-322 in Biological Control in Crop Production. Beltsville Symp. Agric. Res. Vol. 5, Ed. Allanheld, Osmun & Co., Totowa, N.J. (1981)).
It would be useful to provide a means of biological control of plant pathogens such as Phytophthora in soil and on plant surfaces using a microorganism capable of exerting its effect in the presence of other competing microorganisms.